Dimerization propensities of Synucleins are not predictive for Synuclein aggregation

Eckermann, Katrin; Kügler, Sebastian; Bähr, Mathias

doi:10.1016/j.bbadis.2015.05.002

Cited by 17 publications

(41 citation statements)

References 63 publications

(77 reference statements)

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“…When expressed individually, Vn-a-syn was expressed at 6-to 16-fold higher levels than a-syn-Vc ( Fig. 2A) and at levels higher than those of any of the other tested constructs (WT-a-syn, a-syn-Vc and a-syn-Venus), suggesting an increased stability or decreased turnover of Vn-a-syn that is consistent with previous observations (Dominguez-Meijide et al, 2020;Eckermann, Kügler, & Bähr, 2015;Gustafsson et al, 2018;Lázaro et al, 2014).…”

Section: Resultssupporting

confidence: 90%

“…2E). Surprisingly, this imbalance has been repeatedly observed but not discussed in previous studies (Cai et al, 2018;Dominguez-Meijide et al, 2020;Eckermann et al, 2015;Gustafsson et al, 2018;Lázaro et al, 2016Lázaro et al, , 2014Prasad et al, 2018;Zondler et al, 2014).…”

Section: Resultsmentioning

confidence: 70%

“…Only one report (Eckermann et al, 2015) suggested that the differential expression of the two constructs could lead to a potential entrapment of a-syn-Vc by the strongly overexpressed…”

Section: Resultsmentioning

confidence: 99%

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Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Frey

AlOkda

Lashuel

2020

Preprint

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Abbreviations:a-syn: a-synuclein; a-syn-V: a-synuclein-full length Venus; Vn-a-syn: n-terminal fragment of Venus fused to the amino-terminus of a-syn; a-syn-Vc: c-terminal fragment of Venus fused to the carboxy-terminus of a-syn; BiFC: bimolecular fluorescent complementation; PD: Parkinson´s disease; SEC: size exclusion chromatography Abstract Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed, including mouse, rat and Drosophila models, based on the assumption that an increase in the fluorescence signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that 1) validate the extent to which the BiFC fluorescent signal correlates with α-syn oligomerization at the biochemical level; 2) provide a structural characterization of the oligomers and aggregates formed by the BiFC fragments; or 3) investigate the extent to which the oligomers by the fluorescent complex resemble oligomers that form on the pathway to α-syn fibrillization. To address this knowledge gap, we first analyzed the expression level and oligomerization properties of the individual constituents of α-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 cells using multiple approaches, including size exclusion chromatography, semiquantitative Western blot analysis, in-cell crosslinking, immunocytochemistry and sedimentation assays. Next, we investigated the biochemical and aggregation properties of α-syn upon co-expression of both BiFC fragments. Our results show that 1) the two BiFC fragments are not expressed at the same level since C-terminal-Venus fused to α-syn (a-syn-Vc) was present in very low abundance; 2) the fragment with Nterminal-Venus fused to α-syn (Vn-a-syn) exhibited a high propensity to form oligomers and higher-order aggregates; 3) the expression of either or both fragments does not result in the formation of α-syn fibrils, cellular inclusions or the accumulation of pS129 immunoreactive αsyn aggregates. Furthermore, our results suggest that only a small fraction of Vn-a-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of a-syn-Vc in Vn-a-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate α-syn oligomerization in cells or in vivo. Altogether, our results suggest that oligomerization, aggregation and cell-tocell transmission assays and model systems based on a-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 70%

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Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Frey

AlOkda

Lashuel

2020

Preprint

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“…The protein has a tendency to dimerize, as detected by AlphaScreen, two-color single-molecule coincidence and brightness analysis. This dimerization propensity has been noted before8182. Formation of oligomers of ~30 proteins follows, that more rarely leads to formation of type II fibrils.…”

Section: Discussionsupporting

confidence: 55%

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

Sierecki

Giles

Bowden

et al. 2016

Sci Rep

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Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

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“…So far, the large majority of detection methods for proteins in vivo is based on the use of antibodies . A good number of techniques have been applied to detect αS self‐interaction using complementation assays or proximity ligations assays or confocal microscopy methods .…”

Section: Alpha‐synuclein and Parkinson's Diseasementioning

confidence: 99%

Shedding light on aberrant interactions – a review of modern tools for studying protein aggregates

et al. 2018

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The link between protein aggregation and neurodegenerative disease is well established. However, given the heterogeneity of species formed during the aggregation process, it is difficult to delineate details of the molecular events involved in generating pathological aggregates from those producing soluble monomers. As aberrant aggregates are possible pharmacological targets for the treatment of neurodegenerative diseases, the need to observe and characterise soluble oligomers has pushed traditional biophysical techniques to their limits, leading to the development of a plethora of new tools capable of detecting soluble oligomers with high precision and specificity. In this review, we discuss a range of modern biophysical techniques that have been developed to study protein aggregation, and give an overview of how they have been used to understand, in detail, the aberrant aggregation of amyloidogenic proteins associated with the two most common neurodegenerative disorders, Alzheimer's disease and Parkinson's disease.

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Dimerization propensities of Synucleins are not predictive for Synuclein aggregation

Cited by 17 publications

References 63 publications

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

Shedding light on aberrant interactions – a review of modern tools for studying protein aggregates

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